The reduced bone density and quality that occur in osteoporosis and the associated fracture risk is a major clinical problem in the aged population. Current therapies are aimed at modulating osteoclastic bone resorption and osteoblastic bone formation to maintain bone mass and until recently the osteocyte was overlooked. However, osteocytes are now known to be key regulators of bone mass through controlling the activity of both osteoblasts and osteoclasts. An exciting new paradigm in cell-cell communication is that extracellular vesicles (EV) (exosomes and microvesicles) may provide a novel mechanism for commun- ication between cells. These are membrane-bound particles shed from cells that carry a cargo of proteins, mRNAs and microRNAs (miRNAs). They dock with a target cell, delivering their cargo and altering the function of the target cell. Using a novel transgenic mouse expressing a membrane-targeted GFP in osteocytes, our preliminary live cell imaging data has shown that embedding osteocytes release EV from their cell body and dendrites. We have isolated EV from osteocyte-like cell lines and shown that they contain protein, mRNA and miRNA cargo, are enriched for sclerostin and contain RANKL. These EV have potent effects on osteoblasts to induce differentiation towards an osteocyte phenotype. Based on these novel observations, the overall hypothesis for the proposed studies is that osteocyte derived extracellular vesicles (EV) provide a novel mechanism for regulation of osteoblast and osteoclast function and potentially for molecular crosstalk with cells at distant sites from bone. This exploratory R21 will test this hypothesis using complimentary in vitro and in vivo approaches. Aim 1 will determine the role of osteocyte EV in regulating osteoblast and osteoclast function. EV will be isolated from osteocyte cell lines and their effects in in vitro assays of osteoblast and osteoclast differentiation determined. Live cell imaging will determine the kinetics of EV uptake in osteoblasts and osteoclasts and siRNA and miRNA hairpin Inhibitors will be used to define the mediators of EV effects. In vivo experiments will be performed in mice to confirm the functional effects of osteocyte EV on bone cells in vivo. Aim 2 will use intravital imaging in mice expressing a membrane targeted GFP in osteocytes and DsRed or TdTomato reporters in osteoblasts and osteoclasts. We will determine the dynamics of osteocyte EV release in vivo, whether the EV are shed into the circulation and/or released from bone matrix during bone resorption. Aim 3 will examine whether osteocyte EV provide a mechanism for signal propagation by transferring gene expression changes induced by stimuli such as PTH and oxidative stress to a nave cell population. Proteomics and miRNA profiling will define the cargo of osteocyte EV and its regulation by PTH. These studies may shift paradigms about cell-cell communication in bone and pave the way for exploiting osteocyte EVs as circulating biomarkers and novel therapeutics for diagnosis and treatment of osteoporosis and other bone diseases.